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| United States Patent |
5,759,556
|
|
Burger
, et al.
|
June 2, 1998
|
Skin care compositions containing certain cyclic aliphatic unsaturated
compounds and retinol or retinyl ester
Abstract
Certain cyclic aliphatic unsaturated compounds which sufficiently inhibit
LRAT or ARAT catalyzed esterification of retinol into inactive retinyl
esters, have the same effect on keratinocytes as retinoic acid. Thus,
effects of the retinol or retinyl esters in combination with these cyclic
compounds are analogous to treatment with retinoic acid.
| Inventors:
|
Burger; Allan Robert (Passaic, NJ);
Iwata; Koichi (Washington Township, NJ);
Granger; Stewart Paton (Paramus, NJ);
Rawlings; Anthony Vincent (Warrington, GB2);
Scott; Ian Richard (Allendale, NJ)
|
| Assignee:
|
Chesebrough-Pond's USA Co., Division of Conopco, Inc. (Greenwich, CT)
|
| Appl. No.:
|
722538 |
| Filed:
|
September 27, 1996 |
| Current U.S. Class: |
424/401; 424/49; 514/725; 514/844; 514/846; 514/937 |
| Intern'l Class: |
A61K 007/48 |
| Field of Search: |
424/401,59,725
514/844,846,937
|
References Cited
U.S. Patent Documents
| 4722939 | Feb., 1988 | Loev et al. | 514/529.
|
| 5536740 | Jul., 1996 | Granger et al. | 514/392.
|
Primary Examiner: Venkat; Jyothsan
Attorney, Agent or Firm: Mitelman; Rimma
Claims
What is claimed is:
1. A skin Care composition comprising:
(a) from about 0.001% to about 10% of retinol;
(b) from about 0.0001% to about 50% of a cyclic aliphatic unsaturated
compound selected from the group consisting of alpha damascone, beta
damascone, delta damascone, isodamascone, damascenone, alpha ionone, beta
ionone, allyl alpha ionone, isobutyl ionone, alpha methyl ionone, gamma
methyl ionone, brahmanol, sandanol, alpha terpineol, lyral, ethyl
saffranate, and mixtures thereof; where in the cyclic aliphatic
unsaturated compound at 100 .mu.M concentration inhibits at least 20% of
LRAT or ARAT catalyzed retinol esterification as measured by an in vitro
microsomal assay, and
(c) a cosmetically acceptable vehicle.
2. A skin care composition comprising:
(a) from about 0.001% to about 10% of retinol;
(b) from about 0.0001% to about 50% of a cyclic aliphatic unsaturated
compound selected from the group consisting of alpha damascone, beta
damascone, delta damascone, isodamascone, damascenone, alpha ionone,
brahmanol, lyral, and mixtures thereof; where in the cyclic aliphatic
compound inhibits at least 40% of LRAT or ARAT catalyzed retinol
esterification and
(c) a cosmetically acceptable vehicle.
3. The composition of claim 1 wherein the cyclic aliphatic unsaturated
compound is selected from the group consisting of .alpha.-damascone,
.beta.-damascone, .delta.-damascone, isodamascone, damascenone, alpha
lonone, brahmanol, lyral, and mixtures thereof.
4. A method of treating a skin condition selected from the group consisting
of dry skin, photodamaged skin, wrinkles, age spots, acne, skin
lightening, psoriasis and atopic dermatosis, the method comprising
applying to the skin the composition of claim 1.
Description
FIELD OF THE INVENTION
The present invention relates to skin care compositions containing certain
cyclic aliphatic unsaturated compounds and retinal or retinyl ester.
BACKGROUND OF THE INVENTION
Retinol (vitamin A) is an endogenous compound which occurs naturally in the
human body and is essential for normal epithelial cell differentiation.
Natural and synthetic vitamin A derivatives have been used extensively in
the treatment of a variety of skin disorders and have been used as skin
repair or renewal agents. Retinoic acid has been employed to treat a
variety of skin conditions, e.g., acne, wrinkles, psoriasis, age spots and
discoloration. See e.g., Vahlquist, A. et al., J. Invest. Dermatol., Vol.
94, Holland D. B. and Cunliffe, W. J. (1990), pp. 496-498; Ellis, C. N. et
al., "Pharmacology of Retinols in Skin", Vasel, Karger, Vol. 3, (1989),
pp.249-252; Lowe, N. J. et al., "Pharmacology of Retinols in Skin", Vol.
3, (1989), pp.240-248; PCT Patent Application No. WO 93/19743.
It is believed that the use of retinol or esters of retinol would be
preferred over retinoic acid. Retinol is an endogenous compound which
occurs naturally in the human body and is essential for normal epithelial
cell differentiation. Retinol is also considered much safer than retinoic
acid. Esters of retinol hydrolyze in-vivo to produce retinol. It is
believed that retinal esters and retinol are metabolically converted in
the skin into retinoic acid according to the following mechanism:
##STR1##
However, most of the endogenously applied retinol is rapidly converted into
inactive fatty esters for storage in epidermal cells (keratinocytes).
Esterification of retinol into inactive retinyl esters is achieved in
cells by transfer of a fatty acyl group from an acyl CoA, catalyzed by the
enzyme acyl CoA retinol transferase (ARAT), or by the transfer of an acyl
group from phosphatidyl choline, catalyzed by the enzyme lecithin retinol
acyl transferase (LRAT). These esterification reactions are very efficient
in keratinocytes--the majority (95%) of cellular retinoids are in the form
of retinyl fatty esters. Thus, unfortunately, although retinol and retinyl
esters are safer to use than retinoic acid, they are less effective than
retinoic acid at providing skin benefits.
The present invention is based, in part, on the discovery that certain
cyclic hydrocarbons inhibit these esterification reactions and thus
potentiate the action of retinol by increasing the amount of retinol
available for conversion to retinoic acid. Thus, a mixture of selected
cyclic hydrocarbons with retinol or retinyl esters mimics retinoic acid
yet is safer to use than retinoic acid.
SUMMARY OF THE INVENTION
The present invention includes, in part, a skin conditioning composition
containing:
(a) from about 0.001% to about 10% of retinol or a retinyl ester;
(b) from about 0.0001% to about 50% of a cyclic aliphatic unsaturated
compound which at 100 .mu.M concentration inhibits at least 20% of LRAT or
ARAT catalyzed retinol esterification as measured by an in vitro
Microsomal Assay; and
(c) a cosmetically acceptable vehicle.
The cyclic aliphatic unsaturated hydrocarbonssuitable for use in the
present invention are aliphatic unsaturated aldehydes, ketones alcohols
and esters.
The term "conditioning" as used herein means prevention and treatment of
dry skin, photodamaged skin, appearance of wrinkles, age spots, aged skin,
increasing stratum corneum flexibility, lightening skin color, controlling
sebum excretion and generally increasing the quality of skin. The
composition may be used to improve skin desquamation and epidermal
differentiation.
The presence of the selected cyclic hydrocarbon in the inventive product
substantially improves the performance of retinol or a retinyl ester.
According to the present invention, by virtue of including an effective
amount of a selected cyclic aliphatic unsaturated compound which at 100
.mu.M concentration inhibits at least 20% of LRAT catalyzed retinol
esterification as measured by in vitro Microsomal Assay, into compositions
containing retinol or a retinyl ester, the performance of the compositions
is substantially improved.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The inventive compositions contain, as a first essential ingredient, a
compound selected from the group consisting of retinol or a retinyl ester.
The term "retinol" includes the following isomers of retinol:
all-trans-retinol, 13-cis-retinol, 11-cis-retinol, 9-cis-retinol,
3,4-didehydro-retinol. Preferred isomers are all-transretinol,
13-cis-retinol, 3,4-didehydro-retinol, 9-cis-retinol. Most preferred is
all-transretinol, due to its wide commercial availability.
Retinyl ester is an ester of retinol. The term "retinol" has been defined
above. Retinyl esters suitable for use in the present invention are
C.sub.1 -C.sub.30 esters of retinol, preferably C.sub.2 -C.sub.20 esters,
and most preferably C.sub.2, C.sub.3, and C.sub.6 esters because they are
more commonly available. Examples of retinyl esters include but are not
limited to: retinyl palmitate, retinyl formate, retinyl acetate, retinyl
propionate, retinyl butyrate, retinyl valerate, retinyl isovalerate,
retinyl hexanoate, retinyl heptanoate, retinyl octanoate, retinyl
nonanoate, retinyl decanoate, retinyl undecandate, retinyl laurate,
retinyl tridecanoate, retinyl myristate, retinyl pentadecanoate, retinyl
heptadeconoate, retinyl stearate, retinyl isostearate, retinyl
nonadecanoate, retinyl arachidonate, retinyl behenate, retinyl linoleate,
retinyl oleate.
The preferred ester for use in the present invention is selected from
retinyl palmitate, retinyl acetate and retinyl propionate, because these
are the most commercially available and therefore the cheapest. Retinyl
linoleate is also preferred due to its efficacy.
Retinol or retinyl ester is employed in the inventive composition in an
amount of from about 0.001% to about 10%, preferably in an amount of from
about 0.01% to about 1%, most preferably in an amount of from about 0.01%
to about 0.5%.
The second essential ingredient of the inventive compositions is a selected
cyclic aliphatic unsaturated compound. The cyclic aliphatic unsaturated
compound suitable for use in the present invention inhibits at 100 .mu.M
concentration, at least 20% of LRAT or ARAT catalyzed retinol
esterification as measured by in vitro Microsomal Assay. The in vitro
Microsomal Assay employed for determining the suitability of the inclusion
of the compound in the inventive compositions is as follows:
In vitro Microsomal Assay:
Microsomes are obtained as described in: J. C. Saari and D. L. Bredberg,
"CoA and Non-CoA Dependent Retinol Esterification in Retinal Figment
Epithelium" J. Biol. Chem. 263, 8084-90 (1988).
A solution containing 0.1M sodium phosphate pH 7 buffer, 5 mM
dithiothreitol, 2 mg/ml bovine serum albumin, 40 micromolar palmitoyl CoA,
40 micromolar dilauroyl phosphatidyl choline, 10 micromolar retinol and a
test compound or a solvent blank, is incubated for 1 hour at 37.degree. C.
with a microsomal fraction isolated from bovine retinal pigment epithelial
cells. After incubation, the reaction was quenched by addition of an equal
volume of ethanol, and the retinyl esters formed (retinyl laurate from the
LRAT catalyzed reaction and retinyl palmitate from ARAT catalyzed
reaction) are extracted with hexane. The hexane layer is removed,
evaporated under nitrogen, and the residue analyzed by HPLC on a
3.9.times.300 mm C.sub.18 reversed phase column using a 80% methanol in
tetrahydrofuran mobile phase and fluorescence detection (325 nm
excitation, 480 nm emission) to quantitate the retinyl ester. The quantity
of ester formed in the presence of the solvent blank is taken as 100%, and
this is used to calculate the percent inhibition of ester formation for
the compounds tested. As a control, an aliquot of microsomes is
inactivated by boiling for 5 minutes, which results in at least 95%
inhibition of ester formation.
In a preferred embodiment of the invention, a cyclic aliphatic unsaturated
compound is selected which, at a 100 .mu.M concentration, inhibits at
least 40% of LRAT or ARAT catalyzed retinol esterification. A preferred
cyclic aliphatic unsaturated compound is selected from cyclic aliphatic
unsaturated aldehydes, ketones, alcohols and esters.
Suitable cyclic aliphatic unsaturated aldehydes, ketones, alcohols and
esters include but are not limited to: alpha damascone, beta damascone,
delta damascone, isodamascone, damascenone, alpha ionone, beta ionone,
allyl alpha ionone, isobutyl ionone, alpha methyl ionone, gamma methyl
ionone, brahmanol, sandanol, alpha terpineol, lyral, ethyl saffranate, and
mixtures thereof. The structures of these compounds are as follows:
##STR2##
Preferably, in order to maximize performance at a minimum cost, a cyclic
aliphatic unsaturated compound is selected from the group consisting of
damascones and ionones having the structures described above.
Most preferably, the cyclic aliphatic unsaturated compound is a
.alpha.-Damascone and/or .alpha.-lonone.
The cyclic aliphatic unsaturated compound is included in the inventive
compositions in an amount ranging from about 0.0001% to about 50%,
preferably from about 0.01% to about 10%, most preferably from about 0.1%
to about 5%.
Cosmetically Acceptable Vehicle
The composition according to the invention also comprises a cosmetically
acceptable vehicle to act as a dilutant, dispersant or carrier for the
active components in the composition, so as to facilitate their
distribution when the composition is applied to the skin.
Vehicles other than water can include liquid or solid emollients, solvents,
humectants, thickeners and powders. An especially preferred nonaqueous
carrier is a polydimethyl siloxane and/or a polydimethyl phenyl siloxane.
Silicones of this invention may be those with viscosities ranging anywhere
from about 10 to 10,000,000 centistokes at 25 .degree. C. Especially
desirable are mixtures of low and high viscosity silicones. These
silicones are available from the General Electric Company under trademarks
Vicasil, SE and SF and from the Dow Corning Company under the 200 and 550
Series. Amounts of silicone which can be utilized in the compositions of
this invention range anywhere from 5 to 95%, preferably from 25 to 90% by
weight of the composition.
The cosmetically acceptable vehicle will usually form from about 5 to about
99.9%, preferably from about 25 to about 80% by weight of the composition,
and can, in the absence of other cosmetic adjuncts, form the balance of
the composition.
Optional Skin Benefit Materials and Cosmetic Adjuncts
An oil or oily material may be present, together with an emulsifier to
provide either a water-in-oil emulsion or an oil-in-water emulsion,
depending largely on the average hydrophilic-lipophilic balance (HLB) of
the emulsifier employed.
Various types of active ingredients may be present in cosmetic compositions
of the present invention. Actives are defined as skin or hair benefit
agents other than emollients and other than ingredients that merely
improve the physical characteristics of the composition. Although not
limited to this category, general examples include sunscreens, tanning
agents.
Sunscreens include those materials commonly employed to block ultraviolet
light. Illustrative compounds are the derivatives of PABA, cinnamate and
salicylate. For example, octyl methoxycinnamate and 2-hydroxy-4-methoxy
benzophenone (also known as oxybenzone) can be used. Octyl
methoxycinnamate and 2-hydroxy-4-methoxy benzophenone are commercially
available under the trademarks, Parsol MCX and Benzophenone-3,
respectively. The exact amount of sunscreen employed in the emulsions can
vary depending upon the degree of protection desired from the sun's UV
radiation.
Another preferred optional ingredient is selected from essential fatty
acids (EFAs), i.e., those fatty acids which are essential for the plasma
membrane formation of all cells, in keratinocytes EFA deficiency makes
cells hyperproliferative. Supplementation of EFA corrects this. EFAs also
enhance lipid biosynthesis of epidermis and provide lipids for the barrier
formation of the epidermis. The essential fatty acids are preferably
chosen from linoleic acid, .gamma.-linolenic acid, homo-.gamma.-linolenic
acid, columbinic acid, eicosa-(n-6,9,13)-trienoic acid, arachidonic acid,
.gamma.-linolenic acid, timnodonic acid, hexaenoic acid and mixtures
thereof.
Emollients are often incorporated into cosmetic compositions of the present
invention. Levels of such emollients may range from about 0.5% to about
50%, preferably between about 5% and 30% by weight of the total
composition. Emollients may be classified under such general chemical
categories as esters, fatty acids and alcohols, polyols and hydrocarbons.
Esters may be mono- or di-esters. Acceptable examples of fatty di-esters
include dibutyl adipate, diethyl sebacate, diisopropyl dimerate, and
dioctyl succinate. Acceptable branched chain fatty esters include
2-ethyl-hexyl myristate, isopropyl stearate and isostearyl palmitate.
Acceptable tribasic acid esters include triisopropyl trilinoleate and
trilauryl citrate. Acceptable straight chain fatty esters include lauryl
palmitate, myristyl lactate, oleyl eurcate and stearyl oleate. Preferred
esters include coco-caprylate/caprate(a blend of cococaprylate and
coco-caprate), propylene glycol myristyl ether acetate, diisopropyl
adipate and cetyl octanoate.
Suitable fatty alcohols and acids include those compounds having from 10 to
20 carbon atoms. Especially preferred are such compounds such as cetyl,
myristyl, palmitic and stearyl alcohols and acids.
Among the polyols which may serve as emollients are linear and branched
chain alkyl polyhydroxyl compounds. For example, propylene glycol,
sorbitol and glycerin are preferred. Also useful may be polymeric polyols
such as polypropylene glycol and polyethylene glycol. Butylene and
propylene glycol are also especially preferred as penetration enhancers.
Exemplary hydrocarbons which may serve as emollients are those having
hydrocarbon chains anywhere from 12 to 30 carbon atoms. Specific examples
include mineral oil, petroleum jelly, squalene and isoparaffins.
Another category of functional ingredients within the cosmetic compositions
of the present invention are thickeners. A thickener will usually be
present in amounts anywhere from 0.1 to 20% by weight, preferably from
about 0.5% to 10% by weight of the composition. Exemplary thickeners are
cross-linked polyacrylate materials available under the trademark Carbopol
from the B. F. Goodrich Company. Gums may be employed such as xanthan,
carrageenan, gelatin, karaya, pectin and locust beans gum. Under certain
circumstances the thickening function may be accomplished by a material
also serving as a silicone or emollient. For instance, silicone gums in
excess of 10 centistokes and esters such as glycerol stearate have dual
functionality.
Powders may be incorporated into the cosmetic composition of the invention.
These powders include chalk, talc, Fullers earth, kaolin, starch, smectite
clays, chemically modified magnesium aluminum silicate, organically
modified montmorillonite clay, hydrated aluminum silicate, fumed silica,
aluminum starch octenyl succinate and mixtures thereof.
Other adjunct minor components may also be incorporated into the cosmetic
compositions. These ingredients may include coloring agents, opacifiers
and perfumes. Amounts of these materials may range anywhere from 0.001% up
to 20% by weight of the composition. The cyclic compounds employed as
actives in the present invention are, in addition to their role in the
present invention, perfumes, and thus may have a dual function inventive
compositions. Additional perfumes may be incorporated.
Use of the Composition
The composition according to the invention is intended primarily as a
product for topical application to human skin, especially as an agent for
conditioning and smoothening the skin, preventing or reducing the
appearance of wrinkled or aged skin, skin lightening and sebum control.
In use, a small quantity of the composition, for example from 1 to 5 ml, is
applied to exposed areas of the skin, from a suitable container or
applicator and, if necessary, it is then spread over and/or rubbed into
the skin using the hand or fingers or a suitable device.
Product Form and Packaging
The topical skin treatment composition of the invention can be formulated
as a lotion or a cream or a gel. The composition can be packaged in a
suitable container to suit its viscosity and intended use by the consumer.
For example, a lotion or fluid cream can be packaged in a bottle or a
roll-ball applicator, or a capsule, or a propellant-driven aerosol device
or a container fitted with a pump suitable for finger operation. When the
composition is a cream, it can simply be stored in a non-deformable bottle
or squeeze container, such as a tube or a lidded jar.
The invention accordingly also provides a closed container containing a
cosmetically acceptable composition as herein defined.
The following specific examples further illustrate the invention, but the
invention is not limited thereto.
MATERIALS AND METHODS
Cell Culture
Human keratinocytes, isolated from neonatal foreskin by trypsin treatment
were grown in Dulbecco Modification Eagle (DME) Hams F12 (1:1) medium/10%
fetal calf serum in the presence of irradiated 3T3 mouse fibroblasts for
establishing dividing keratinocyte colonies. Cells were grown under the
above condition until their second passage and kept frozen for future use.
Frozen second passage keratinocytes were thawed and plated into the above
medium and grown for five days before they were switched to a serum-free
MCDB 153-based medium keratinocyte growth medium (KGM) from Clonetics
Corporation, San Diego, Calif., containing 0.15 mM Ca, or keratinocyte
serum-free media (KSFM) from GIBCO containing 0.09 mM Ca). On day 7, when
the cells were 80-90% confluent, they were trypsinized and plated in the
serum-free medium for the various experiments.
Thymidine Assay
.sup.3 H-Thymidine Incorporation and Keratinocyte Proliferation
The incorporation of .sup.3 H-thymidine by cultured keratinocytes was used
as an assay of keratinocyte proliferation. Thymidine is one of four
deoxynucleosides which are the monomeric units of DNA, the universal
library of genetic information in the animal kingdom. Prior to cell
division of a somatic cell such as a keratinocyte, the complete genome of
the cell undergoing cell division is replicated. This involves large scale
DNA synthesis by the cell and enables both daughter cells to receive
identical copies of the genetic material. When .sup.3 H-thymidine is
included in the culture media of keratinocytes which are synthesizing DNA
in preparation for cell division then the labelled nucleoside is
incorporated into the newly synthesized DNA. The extent of incorporation
of .sup.3 H-thymidine into a population of cells is proportional to the
rate of DNA synthesis by this population of cells and therefore an
indication of their cellular proliferation.
Keratinocytes (that were cultured as described above) were plated in 24
well plates at a density of about 20,000 cells per well in 1 ml media.
After incubation for four days or until the cells were 60-70% confluent,
the media was changed. Test compounds were added (in triplicate) to the
wells 24 hours after the media change, and four hours later 1.mu.Ci .sup.3
H-Thymidine in 50 .mu.l media was added per well. Cells were incubated for
a further 24 hours. Media was removed from the cells, 10% ice cold
trichloroacetic acid (TCA) added and plates were incubated on ice for 30
minutes. Cells were washed five times with 5% TCA and allowed to dissolve
in 500 .mu.l 0.1M NaOH for at least one hour (usually overnight). The
preparations were neutralized with 0.1M HCl; 50 .mu.l of the cell
preparation was used to determine total protein content. Disintegrations
per minute (DPM) from .sup.3 H labelling of DNA was determined by liquid
scintillation counting of 900 .mu.l of the cell preparation. Thymidine
incorporation results were expressed as DPM/.mu.g protein.
TRANSGLUTAMINASE ASSAY
Transglutaminase Assay and Keratinocyte Differentiation
During the process of terminal differentiation in the epidermis, a 15 nm
thick layer of protein, known as the cornified envelope (CE) is formed on
the inner surface of the cell periphery. The CE is composed of numerous
distinct proteins which have been cross-linked together by the formation
of N .sup..epsilon. -(.gamma.-glutamyl) lysine isodipeptide bonds
catalyzed by the action of at least two different transglutaminases
(TGases) expressed in the epidermis. TGase I is expressed in abundance in
the differentiated layers of the epidermis, especially the granular layer,
but is absent in the undifferentiated basal epidermis. Thus TGase I is a
useful marker of epidermal keratinocyte differentiation with high TGase I
levels indicating a more differentiated state. An ELISA based TGase I
assay, using a TGase I antibody, was used to assess the state of
differentiation of the cultured keratinocytes in the examples that follow.
For Example 1, the following procedure was used:
Keratinocytes (cultured as described above) were plated in 96 well plates
at a density of 3,000 cells per well in 200 .mu.l media. After incubation
for four days the media was changed to media containing test compounds
(six replicates per test). The cells were cultured for a further 72 hours
after which time the media was aspirated and the plates stored at
-70.degree. C. Plates were removed from the freezer, and the cells washed
with PBS. 100 .mu.l sterile water was added and the cells were freeze
fractured by freezing at -70.degree. C. then thawing. The cells were
incubated for one hour at room temperature (R/T) with PBS/3% BSA (wash
buffer, bovine serum albumin), then rinsed with a fresh aliquot of wash
buffer. Cells were incubated with 50 .mu.l of primary antibodies
monoclonal anti-human transglutaminase mouse antibody (IgG) obtained from
Biomedical Industries diluted 1:2,000 in wash buffer for one hour,
37.degree. C. then rinsed two times with wash buffer. Cells were then
incubated with 50 .mu.l of secondary antibody (Fab fragment, peroxidase
conjugated anti-mouse IgG obtaining from Amersham) diluted 1:4,000 in wash
buffer for one hour at 37.degree. C., then rinsed two times with wash
buffer. Cells were incubated with substrate solution (4 mg o-phenylene
diamine and 3.3 .mu.l 30% H.sub.2 O.sub.2 in 10 ml 0.1M citrate buffer pH
5.0) for five minutes, R/T, in darkness (under aluminum foil). The
reaction was stopped by the addition of 50 .mu.l 4N H.sub.2 SO.sub.4. The
absorbance of samples was read at 492 nm in the plate reader. Out of the
six replicates, four were treated with both antibodies, two were treated
only with the secondary antibody (i.e., to determine background binding of
enzyme conjugated Ab). TGase levels were determined by subtracting
background from the readings from each treatment and determining
mean.+-.s.d. for the replicates exposed to both Ab.
For Example 4, the following procedure was used:
Keratinocytes (cultured as described above) were plated in 96 well plates
at a density of 3,000 cells per well in 200 .mu.l of cell culture media.
After incubation for four days, the media was changed to media containing
test compounds (six replicates per test). The cells were cultured for a
further 72 hours after which time the media was aspirated and the plates
stored at -70.degree. C. After the plates were removed from the freezer,
the cells were further freezed fractured by freezing and thawing and then
washed 3.times. with PBS. The cells were incubated for one hour at room
temperature (R/T) with TBS/5% BSA buffer. Cells were then incubated with
100 .mu.l of monoclonal anti-human transglutaminase (IgG) mouse antibody
(primary antibody) obtained from Biomedical Technologies Inc. diluted
1:2000 in TBS/1% BSA buffer for two hours at 37.degree. C., and then
rinsed six times with wash buffer (TBS/1% BSA/0.05% Tween-20). Cells were
next incubated with 100 .mu.l of Fab fragment, peroxidase conjugated
anti-mouse IgG antibody (secondary antibody) from Amersham diluted 1:4,000
in wash buffer for two hours at 37.degree. C. and then rinsed three times
with wash buffer and three times with PBS. Cells were incubated with
substrate solution (4 mg o-phenylene diamine and 3.3 .mu.l 30% H.sub.2
O.sub.2 in 10 mL 0.1M citrate buffer, pH 5.0) for five minutes at R/T and
in darkness (under aluminum foil). The reaction was stopped by the
addition of 50 .mu.l 4N H2SO4. The absorbance of samples was read at 492
nm in the plate reader. Out of the six replicates, four were treated with
both antibodies, two were treated only with the secondary antibody (i.e.,
to determine the background binding of the enzyme conjugated antibody).
Transglutaminase I levels were determined by subtracted background from
the readings from each treatment and determining the mean s.d. for the
replicates exposed to both antibodies.
DNA Assay
The level of TGase-1 detected after treatment of the cells could be
influenced by cell number, i.e., the greater the number of cells the
greater the level of TGase-1 detected. The level of TGase-1 was normalized
to DNA content of the cells in the same well thus eliminating variation
due to differences in cell number. DNA quantitation is a particularly
useful indicator of cell number, including keratinocyte cell number,
because each cell has to all intents and purposes an identical genome and
therefore an identical quantity of DNA. The total DNA content of a well of
cells therefore is directly proportional to the cell number in that well.
Quantitation of DNA was used to normalize the TGase data to cell number.
Keratinocytes were plated in 96 well plates at a density of 3,000 cells per
well in 200 .mu.l media. After incubation for four days the media was
changed for media containing test compounds (6 replicates per test). The
cells were cultured for a further 72 hours after which time the media was
aspirated and the plates stored for at least 1.5 hours at -70.degree. C.
Plates were removed from the freezer and thawed for 30 minutes. 100
.mu.l/well of Hoechst dye (1 .mu.g/ml final concentration) was added and
this was incubated for 15 minutes, covered and then read in a fluorimeter
(ex. 360 nm and em. 460 nm). The dye solution was removed and the wells
were rinsed with PBS in preparation for the TGase assay.
EXAMPLE 1
Retinoic acid is more effective than retinol at altering keratinocyte
differentiation state
A. The effect on incorporation of .sup.3 H-thymidine .mu.g soluble protein
24 hours after the addition of retinoic acid or retinol at various
concentrations was examined. The results that were obtained are summarized
in Table 1A.
TABLE 1A
__________________________________________________________________________
Effect of Retinoic Acid (RA) and
Retinol (ROH) on Keratinocyte Thymidine Incorporation
mean Thymidine
incorp./.mu.g
protein .+-. s.d
p value vs
p value vs
p value vs
p value vs
Treatment
(% control)
Control
10.sup.-7 M ROH
10.sup.-8 M ROH
10.sup.-9 M ROH
__________________________________________________________________________
Control 2094 .+-. 140 (100%)
-- 0.202 0.501 0.203
2.5 .times. 10.sup.-7 M RA
2475 .+-. 116 (118%)
0.005
0.032 0.004 0.002
2.5 .times. 10.sup.-7 M ROH
2218 .+-. 73 (106%)
0.202
-- 0.021 0.005
2.5 .times. 10.sup.-8 M RA
2686 .+-. 72 (128%)
0.001
0.001 0.001 0.001
2.5 .times. 10.sup.-8 M ROH
2034 .+-. 46 (97%)
0.501
0.021 -- 0.121
2.5 .times. 10.sup.-9 M RA
2556 .+-. 80 (122%)
0.001
0.006 0.001 0.001
2.5 .times. 10.sup.-9 M ROH
1977 .+-. 19 (94%)
0.203
0.005 0.121 --
__________________________________________________________________________
n = 3
All concentrations of retinoic acid tested, i.e., 2.5.times.10.sup.-7 M,
2.5.times.10.sup.-8 and 2.5.times.10.sup.-9 M, significantly increased
keratinocyte proliferation over both the ethanol control and each of the
2.5.times.10.sup.-7 M, 2.5.times.10.sup.-8 M and 2.5.times.10.sup.-9 M
retinol treatments and they did so in a dose dependant manner. This is
consistent with retinoic acid having a greater stimulatory effect on
epithelial proliferation than retinol.
B. The effect on Transglutaminase levels normalized to DNA content of the
cells after addition of retinoic acid and retinol was examined and the
results are shown in Table 1B.
TABLE 1B
__________________________________________________________________________
mean TGase/
DNA .times. 10.sup.-4 .+-. s.d
p value vs
p value vs
p value vs
p value vs
Treatment
(% control)
Control
10.sup.-7 ROH
10.sup.-8 ROH
10.sup.-9 ROH
__________________________________________________________________________
Control 2.44 .+-. 0.24 (100%)
-- 0.001 0.001 0.001
2.5 .times. 10.sup.-7 M RA
0.16 .+-. 0.11 (7%)
0.001
0.001 0.001 0.001
2.5 .times. 10.sup.-7 M ROH
1.14 .+-. 0.22 (47%)
0.001
-- 0.001 0.001
2.5 .times. 10.sup.-8 M RA
1.34 .+-. 0.40 (55%)
0.001
0.001 0.001 0.001
2.5 .times. 10.sup.-8 M ROH
1.89 .+-. 0.30 (77%)
0.001
0.001 -- 0.001
2.5 .times. 10.sup.-9 M RA
1.87 .+-. 0.49 (77%)
0.001
0.001 0.784 0.001
2.5 .times. 10.sup.-9 M ROH
2.70 .+-. 0.59 (>100%)
0.001
0.001 0.001 --
__________________________________________________________________________
n = 3
All concentrations of retinoic acid tested, i.e., 2.5.times.10.sup.-7 M,
2.5.times.10.sup.-8 M and 2.5.times.10.sup.-9 M decreased keratinocyte
differentiation over both the ethanol control and did so to a
significantly greater extent than each of the corresponding
2.5.times.10.sup.-7 M, 2.5.times.10.sup.-8 M and 2.5.times.10.sup.-9 M
retinol treatments. The decrease in transglutaminase level was dose
dependent for both retinoic acid and retinol. This is consistent with
retinoic acid having a greater inhibitory effect on epithelial
differentiation than retinol.
EXAMPLE 2
In vitro microsomal esterification of retinol:
Microsomes are obtained as described in: J. C. Saari and D. L. Bredberg,
"CoA and Non-CoA Dependent Retinol Esterification in Retinal Pigment
Epithelium" J. Biol. Chem. 23, 8084-90 (1988).
A solution containing 0.1M sodium phosphate pH 7 buffer, 5 mM
dithiothreitol, 2 mg/ml bovine serum albumin, 40 micromolar palmitoyl CoA,
40 micromolar dilauroyl phosphatidyl choline, 10 micromolar retinol and a
test compound or solvent blank, was incubated for 1 hour at 37.degree. C.
with a microsomal fraction isolated from bovine retinal pigment epithelial
cells. After incubation, the reaction was quenched by addition of an equal
volume of ethanol, and the retinyl esters formed (retinyl palmitate from
the ARAT catalyzed reaction, and retinyl laurate from the LRAT catalyzed
reaction) were extracted with hexane. The hexane layer was removed,
evaporated under nitrogen, and the residue analyzed by HPLC on a
3.9.times.300 mm C18 reversed phase column using a 80% methanol in
tetrahydrofuran mobile phase and fluorescence detection (325 nm
excitation, 480 nm emission) to quantitate the retinyl esters. The
quantity of ester formed in the presence of the solvent blank was taken as
100%, and this was used to calculate the percent inhibition of ester
formation for the compounds tested. As a control, an aliquot of microsomes
was inactivated by boiling for 5 minutes, which resulted in at least 95%
inhibition of ester formation.
The results that were obtained are summarized in Tables A and B.
The compounds in Table A were tested at a 100 .mu.M concentration. The
compounds in Table B were tested at a 10 .mu.M concentration.
TABLE A
______________________________________
% INHIBITION,
COMPOUND % INHIBITION, ARAT
LRAT
______________________________________
alpha damascone
83 98
beta damascone
84 92
delta damascone
87 95
isodamascone 80 92
damascenone 70 79
alpha ionone 45 49
beta ionone 22 24
allyl alpha ionone
22 36
isobutyl ionone
8 45
alpha methyl ionone
67 77
gamma methyl ionone
21 38
brahmanol 70 75
sandanol 15 43
alpha terpineol
26 25
timberol 34 33
lyral 76 71
tonalid 50 33
ethyl saffranate
51 49
traseolide 41 21
______________________________________
TABLE B
______________________________________
% INHIBITION,
COMPOUND % INHIBITION, ARAT
LRAT
______________________________________
alpha damascone
67 87
beta damascone
45 52
delta damascone
58 64
damascenone 23 29
allyl alpha ionone
16 17
______________________________________
It can be seen from the results in Tables A and B that certain cyclic
aliphatic unsaturated compounds are potent inhibitors of LRAT and ARAT
catalyzed retinol esterification.
COMPARATIVE EXAMPLE 3
Example 2 was repeated with additional cyclic aliphatic unsaturated
compounds. The results that were obtained are summarized in Table C.
The compounds in Table C were tested at a 100 .mu.M concentration.
TABLE C
______________________________________
% INHIBITION,
COMPOUND % INHIBITION, ARAT
LRAT
______________________________________
dihydro alpha ionone
13 18
alpha ionol 0 0
beta ionol 0 0
cinnamaldehyde
0 0
vanillin 0 0
eucalyptol 0 0
menthol 0 0
thymol 0 0
carvone 0 0
camphor 0 0
mentone 0 0
fenchyl alcohol
12 4
isocyclogeraniol
18 16
sandalone 23 12
dimethyl ionone
0 9
delta methyl ionone
0 10
______________________________________
It can be seen from the results in Table C that not all cyclic aliphatic
unsaturated compounds inhibit or sufficiently inhibit LRAT and ARAT
catalyzed retinol esterification.
EXAMPLE 4
The effect on keratinocyte differentiation of compounds and combinations
listed in Table D was examined. The results were expressed as % of
control. Transglutaminase level was normalized to DNA. Data are from two
experiments where the retinol concentration was changed. The results that
were obtained are summarized in Table D.
TABLE D
______________________________________
CONCENTRATION %
EXPERIMENT #
TREATMENT mM CONTROL
______________________________________
1 Retinoic acid
.00025 24
1 Retinol .001 67
1 .alpha.-Damascone
1 92
1 Retinol + .001 + 1 34
.alpha.-Damascone
2 Retinoic acid
.00025 8
2 Retinol .00025 63.5
2 .alpha.-Damascone
1 86
2 Retinol + .00025 + 1 30
.alpha.-Damascone
______________________________________
The results in Table D show that while .alpha.-Damascone alone and retinol
alone were not very effective, the combination of the two attained
synergistic reduction in transglutaminae mimicking the effect of retinoic
acid on keratinocyte differentiation. This example also establishes a good
correlation between microsomal assay and cell culture data.
EXAMPLE 5
This example illustrates a high internal phase water-in-oil emulsion
incorporating the inventive composition.
______________________________________
% w/w
______________________________________
Retinol 0.5
Fully hydrogenated coconut oil
3.9
.alpha.-Damascone 5
Brij 92* 5
Bentone 38 0.5
MgSO.sub.4 7H.sub.2 O
0.3
Butylated hydroxy toluene
0.01
Perfume qs
Water to 100
______________________________________
*Brij 92 is polyoxyethylene (2) oleyl ether
EXAMPLE 6
This example illustrates an oil-in-water cream incorporating the inventive
composition.
______________________________________
% w/w
______________________________________
Retinol 0.15
Mineral oil 4
.alpha.-Ionone 2
Brij 56* 4
Alfol 16RD* 4
Triethanolamine 0.75
Butane-1,3-diol 3
Xanthan gum 0.3
Butylated hydroxy toluene
0.01
Water to 100
______________________________________
*Brij 56 is cetyl alcohol POE (10)
Alfol 16RD is cetyl alcohol
EXAMPLE 7
This example illustrates an alcoholic lotion incorporating the composition
according to the invention.
______________________________________
% w/w
______________________________________
Retinyl palmitate 0.15
.alpha.-methylionone
0.5
Ethanol 40
Butylated hydroxy toluene
0.01
Water to 100
______________________________________
EXAMPLE 8
This example illustrates another alcoholic lotion containing the inventive
composition.
______________________________________
% w/w
______________________________________
Retinol 0.15
Lyral 0.2
Ethanol 40
Antioxidant
0.1
Water to 100
______________________________________
EXAMPLE 9
This example illustrates a suncare cream incorporating the composition of
the invention:
______________________________________
% w/w
______________________________________
Retinol 0.01
Isodamascone 0.3
Silicone oil 200 cts 7.5
Glycerylmonostearate 3
Cetosteryl alcohol 1.6
Polyoxyethylene-(20)-cetyl alcohol
1.4
Xanthan gum 0.5
Parsol 1789 1.5
Octyl methoxycinnate (PARSOL MCX)
7
Perfume qs
Color qs
Water to 100
______________________________________
EXAMPLE 10
This example illustrates a non-aqueous skin care composition incorporating
the inventive combination.
______________________________________
% w/w
______________________________________
Retinyl palmitate 0.15
.alpha.-Damascone 1
Silicone gum SE-30.sup.1
10
Silicone fluid 345.sup.2
20
Silicone fluid 344.sup.3
55.79
Squalene 10
Linoleic acid 0.01
Cholesterol 0.03
2-hydroxy-n-octanoic acid
0.7
Vitamin E linoleate
0.5
Herbal oil 0.5
Ethanol 2
______________________________________
.sup.1 A dimethyl silicone polymer having a molecular weight of at least
50,000 and a viscosity of at least 10,000 centistokes at 25.degree. C.,
available from GEC
.sup.2 Dimethyl siloxane cyclic pentamer, available from Dow Corning Corp
.sup.3 Dimethyl siloxane tetramer, available from Dow Corning Corp.
Materials employed in the present invention are obtained from the following
sources:
Palmitoyl CoA, BSA, dilauroylphospatidyl choline, retinol, retinoic acid
dithiothreitol--from Sigma
Cyclic aliphatic unsaturated compounds:
Damascones from Firmenich;
lonones from IFF;
Others from suppliers listed in "Flavor and Fragrance Materials" 1991, by
Allured Pub. Co.
It should be understood that the specific forms of the invention herein
illustrated and described are intended to be representative only. Changes,
including but not limited to those suggested in this specification, may be
made in the illustrated embodiments without departing from the clear
teachings of the disclosure. Accordingly, reference should be made to the
following appended claims in determining the full scope of the invention.
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